U.S. patent application number 10/026672 was filed with the patent office on 2003-01-30 for compositions and agents for modulating cellular proliferation and angiogenesis.
Invention is credited to Goldberg, Itzhak D., Pillarisetti, Sivaram.
Application Number | 20030022924 10/026672 |
Document ID | / |
Family ID | 46280213 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022924 |
Kind Code |
A1 |
Pillarisetti, Sivaram ; et
al. |
January 30, 2003 |
Compositions and agents for modulating cellular proliferation and
angiogenesis
Abstract
The invention is directed to pharmaceutical compositions
containing small organic molecules having the ability to mimic or
agonize hepatocyte growth factor/scatter factor (HGF/SF) activity,
useful for promoting, for example, vascularization of tissues or
organs for promoting wound or tissue healing, or augmenting or
restoring blood flow to ischemic tissues such as the heart
following myocardial infarction.
Inventors: |
Pillarisetti, Sivaram;
(Norcross, GA) ; Goldberg, Itzhak D.; (Englewood,
NJ) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
46280213 |
Appl. No.: |
10/026672 |
Filed: |
December 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10026672 |
Dec 19, 2001 |
|
|
|
09896832 |
Jun 29, 2001 |
|
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Current U.S.
Class: |
514/378 ;
514/406; 514/407 |
Current CPC
Class: |
A61K 31/42 20130101;
A61K 31/415 20130101 |
Class at
Publication: |
514/378 ;
514/406; 514/407 |
International
Class: |
A61K 031/42; A61K
031/415 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising an effective HGF/SF
activity modulating amount of a compound with the general formula
I: 6wherein R1 is SO.sub.2Alkyl, wherein alkyl is a C1 to C4
straight-chain, branched or cycloalkyl group; SO.sub.2-Aryl,
wherein aryl is halo, C1 to C4 alkyl- or alkyloxy-substituted
phenyl; COAlkyl, wherein alkyl is C1 to C6 straight-chained alkyl,
branched alkyl or cycloalkyl; COAryl, wherein Aryl is phenyl
substituted with halo, C1-C4 alkyl or alkyloxy; CONHAlkyl wherein
alkyl is C1 to C6 straight-chained alkyl, branched alkyl or
cycloalkyl; or CONHAryl, wherein aryl is phenyl substituted with
halo, C1 to C4 alkyl or C1 to C4 alkyloxy; and R3 is
CHCH-heteroaryl, where in heteroaryl is cis or trans
CHCH-3-thienyl, CHCH-2-furyl, CHCH-3-furyl, substituted
CHCH-thienyl, or CHCH-furyl; phenoxyphenyl; heteroaryl; or aryl
substituted heteroaryl; and a pharmaceutically-acceptable carrier,
excipient or diluent.
2. The pharmaceutical composition of claim 1 wherein R1 is
SO.sub.2CH.sub.3; CO-t-Butyl, or CONHCH.sub.3 and R3 is
CHCH-2-thienyl.
3. The pharmaceutical composition of claim 1 wherein said compound
is
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone;
1-(methylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole;
2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-yl)propan-1-one;
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamide;
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-1-yl)methanone;
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-pyrazol--
1-yl)methanone;
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-pyrazol-1--
yl)methanone;
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-1H-p-
yrazol-1-yl)methanone;
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carb- oxamide;
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thia-
zol-5-yl)-1H-pyrazol-1-yl)methanone;
(3-benzhydryl-1H-pyrazol-1-yl)(4-chlo- rophenyl)methanone;
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-
-1-carboxamide;
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-
-pyrazol-1-yl)methanone;
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl-
)phenoxy)benzonitrile; or
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vin-
yl)-1H-pyrazole.
4. A compound with the general formula I: 7wherein R1 is
SO.sub.2Alkyl, wherein alkyl is a C1 to C4 straight-chain, branched
or cycloalkyl group; SO.sub.2-Aryl, wherein aryl is halo, C1 to C4
alkyl- or alkyloxy-substituted phenyl; COAlkyl, wherein alkyl is C1
to C6 straight-chained alkyl, branched alkyl or cycloalkyl; COAryl,
wherein Aryl is phenyl substituted with halo, C1-C4 alkyl or
alkyloxy; CONHAlkyl wherein alkyl is C1 to C6 straight-chained
alkyl, branched alkyl or cycloalkyl; or CONHAryl, wherein aryl is
phenyl substituted with halo, C1 to C4 alkyl or C1 to C4 alkyloxy;
and R3 is CHCH-heteroaryl, where in heteroaryl is cis or trans
CHCH-3-thienyl, CHCH-2-furyl, CHCH-3-furyl, substituted
CHCH-thienyl, or CHCH-furyl; phenoxyphenyl; heteroaryl; or aryl
substituted heteroaryl.
5. The compound of claim 4 wherein R1 is SO.sub.2CH.sub.3;
CO-t-Butyl, or CONHCH.sub.3 and R3 is CHCH-2-thienyl.
6. The compound of claim 4 wherein said compound is
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone;
1-(methylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole;
2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-yl)propan-1-one;
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamide;
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-1-yl)methanone;
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-pyrazol--
1-yl)methanone;
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-pyrazol-1--
yl)methanone;
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-1H-p-
yrazol-1-yl)methanone;
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carb- oxamide;
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thia-
zol-5-yl)-1H-pyrazol-1-yl)methanone;
(3-benzhydryl-1H-pyrazol-1-yl)(4-chlo- rophenyl)methanone;
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-
-1-carboxamide;
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-
-pyrazol-1-yl)methanone;
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl-
)phenoxy)benzonitrile; or
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vin-
yl)-1H-pyrazole.
7. A method for prophylaxis or treatment of an ischemic condition
or disease in a mammal comprising administering to said mammal an
effective amount of a pharmaceutical composition comprising a
compound of Formula I: 8wherein R1 is SO.sub.2Alkyl, wherein alkyl
is a C1 to C4 straight-chain, branched or cycloalkyl group;
SO.sub.2-Aryl, wherein aryl is halo, C1 to C4 alkyl- or
alkyloxy-substituted phenyl; COAlkyl, wherein alkyl is C1 to C6
straight-chained alkyl, branched alkyl or cycloalkyl; COAryl,
wherein Aryl is phenyl substituted with halo, C1-C4 alkyl or
alkyloxy; CONHAlkyl wherein alkyl is C1 to C6 straight-chained
alkyl, branched alkyl or cycloalkyl; or CONHAryl, wherein aryl is
phenyl substituted with halo, C1 to C4 alkyl or C1 to C4 alkyloxy;
and R3 is CHCH-heteroaryl, where in heteroaryl is cis or trans
CHCH-3-thienyl, CHCH-2-furyl, CHCH-3-furyl, substituted
CHCH-thienyl, or CHCH-furyl; phenoxyphenyl; heteroaryl; or aryl
substituted heteroaryl.
8. The method of claim 7 wherein R1 is SO.sub.2CH.sub.3;
CO-t-Butyl, or CONHCH.sub.3 and R3 is CHCH-2-thienyl.
9. The method of claim 7 wherein said compound is
(4-chlorophenyl)[3-(2-(2-
-thienyl)vinyl)-1H-pyrazol-1-yl]methanone;
1-(methylsulfonyl)-3-(2-(2-thie- nyl)vinyl)-1H-pyrazole;
2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-
-1-yl)propan-1-one;
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxam- ide;
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-1-yl)methanone;
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-pyrazol--
1-yl)methanone;
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-pyrazol-1--
yl)methanone;
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-1H-p-
yrazol-1-yl)methanone;
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carb- oxamide;
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thia-
zol-5-yl)-1H-pyrazol-1-yl)methanone;
(3-benzhydryl-1H-pyrazol-1-yl)(4-chlo- rophenyl)methanone;
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-
-1-carboxamide;
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-
-pyrazol-1-yl)methanone;
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl-
)phenoxy)benzonitrile; or
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vin- yl)-1H-pyrazole.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of Ser. No.
09/896,832, filed Jun. 29, 2001, which is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Scatter factor (SF; also known as hepatocyte growth factor
[HGF], and hereinafter referred to and abbreviated as HGF/SF) is a
pleiotropic growth factor that stimulates cell growth, cell
motility, morphogenesis and angiogenesis. HGF/SF is produced as an
inactive monomer (.about.100 kDa) which is proteolytically
converted to its active form. Active HGF/SF is a heparin-binding
heterodimeric protein composed of a 62 kDa .alpha. chain and a 34
kDa .beta. chain. HGF/SF is a potent mitogen for parenchymal liver,
epithelial and endothelial cells (Matsumoto, K, and Nakamura, T.,
1997, Hepatocyte growth factor (HGF) as a tissue organizer for
organogenesis and regeneration. Biochem. Biophys. Res. Commun. 239,
639-44; Boros, P. and Miller, C. M., 1995, Hepatocyte growth
factor: a multifunctional cytokine. Lancet 345, 293-5). It
stimulates the growth of endothelial cells and also acts as a
survival factor against endothelial cell death (Morishita, R,
Nakamura, S, Nakamura, Y, Aoki, M, Moriguchi, A, Kida, I, Yo, Y,
Matsumoto, K, Nakamura, T, Higaki, J, Ogihara, T, 1997, Potential
role of an endothelium-specific growth factor, hepatocyte growth
factor, on endothelial damage in diabetes. Diabetes 46:138-42).
[0003] HGF/SF synthesized and secreted by vascular smooth muscle
cells stimulate endothelial cells to proliferate, migrate and
differentiate into capillary-like tubes in vitro (Grant, D. S,
Kleinman, H. K., Goldberg, I. D., Bhargava, M. M., Nickoloff, B.
J., Kinsella, J. L., Polverini, P., Rosen, E. M., 1993, Scatter
factor induces blood vessel formation in vivo. Proc. Natl. Acad.
Sci. USA 90:1937-41; Morishita, R., Nakamura, S., Hayashi, S.,
Taniyama, Y., Moriguchi, A., Nagano, T., Taiji, M., Noguchi, H.,
Takeshita, S., Matsumoto, K., Nakamura, T., Higaki, J., Ogihara,
T., 1999, Therapeutic angiogenesis induced by human recombinant
hepatocyte growth factor in rabbit hind limb ischemia model as
cytokine supplement therapy. Hypertension 33:1379-84).
HGF/SF-containing implants in mouse subcutaneous tissue and rat
cornea induce growth of new blood vessels from surrounding tissue.
HGF/SF protein is expressed at sites of neovascularization
including in tumors (Jeffers, M., Rong, S., Woude, G. F., 1996,
Hepatocyte growth factor/scatter factor-Met signaling in
tumorigenicity and invasion/metastasis. J. Mol. Med. 74:505-13;
Moriyama, T., Kataoka, H., Koono, M., Wakisaka, S., 1999,
Expression of hepatocyte growth factor/scatter factor and its
receptor c-met in brain tumors: evidence for a role in progression
of astrocytic tumors Int. J. Mol. Med. 3:531-6). These findings
suggest that HGF/SF plays a significant role in the formation and
repair of blood vessels under physiologic and pathologic
conditions. Further discussion of angiogenic proteins may be found
in U.S. Pat. Nos. 6,011,009 and 5,997,868, both of which are
incorporated herein by reference in their entireties.
[0004] Modulation of cellular proliferation by exogenously-supplied
therapeutic agents has been offered as a new approach for the
prophylaxis and/or treatment of various conditions and diseases in
which limited cellular proliferation is responsible for pathology,
or at least for the prolongation of rebound from a pathological
state to homeostasis. For example, the duration of wound healing,
normalization of myocardial perfusion as a consequence of chronic
cardiac ischemia or myocardial infarction, development or
augmentation of collateral vessel development after vascular
occlusion or to ischemic tissues or organs, and vascularization of
grafted or transplanted tissues, organs, or wound healing, may be
accelerated by promoting cellular proliferation, particularly of
vascular cells.
[0005] It is toward compounds with HGF/SF-like activity and
pharmaceutical compositions comprising them for the prophylaxis and
treatment of various conditions and diseases benefiting from HGF/SF
activity that the present invention is directed.
[0006] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
SUMMARY OF THE INVENTION
[0007] The present invention is directed generally to compositions
and pharmaceutical compositions comprising effective amounts of
compounds that modulate hepatocyte growth factor/scatter factor
(HGF/SF) activities in a mammal, the compounds being useful for the
prophylaxis or treatment of any of a number of conditions or
diseases in which HGF/SF has a therapeutically useful role. The
compounds of the invention generally exhibit HGF/SF stimulatory or
agonist activity.
[0008] The invention is directed to pharmaceutical compositions
comprising a compound that modulates HGF/SF activity with the
general formula I: 1
[0009] wherein
[0010] R1 is SO.sub.2Alkyl, SO.sub.2-Aryl, CO-t-Butyl, COAryl,
CONHAlkyl; or CONHAryl; and
[0011] R3 is CHCH-heteroaryl; phenoxyphenyl; heteroaryl; or Aryl
substituted heteroaryl;
[0012] and a pharmaceutically-acceptable carrier, excipient or
diluent.
[0013] Non-limiting examples of compounds of Formula I include
[0014]
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone;
[0015] 1-(methylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole;
[0016]
2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-yl)propan-1-on-
e;
[0017]
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamide;
[0018]
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-1-yl)methanon-
e;
[0019]
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-py-
razol-1-yl)methanone;
[0020]
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-pyrazol-1-yl)methan-
one;
[0021]
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-1H-pyrazol--
1-yl)methanone;
[0022]
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamide;
[0023]
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thiazo-
l-5-yl)-1H-pyrazol-1-yl)methanone;
[0024] (3-benzhydryl-1H-pyrazol-1-yl)(4-chlorophenyl)methanone;
[0025]
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamid-
e;
[0026]
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-pyrazol--
1-yl)methanone;
[0027]
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl)phenoxy)benzonitr-
ile; and
[0028]
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole.
[0029] The compounds comprising the pharmaceutical compositions of
the invention have been found to mimic or agonize the biological
activities of HGF/SF, and thus are useful in the prophylaxis or
treatment, for example, of conditions or diseases in which enhanced
cellular or vascular proliferation is desirable, among other
desirable activities of HGF/SF. Such conditions or diseases include
hepatic disease, renal disease, bone regeneration dysfunction, poor
hair growth, wound or tissue healing, treatment of various ischemic
diseases, including but not limited to augmenting or restoring
blood flow to ischemic tissues such as the heart following
myocardial infarction as well as in diabetes-related ischemic
conditions. The compounds are also useful in the treatment of
atherosclerosis, including reduction of lipid accumulation in the
vasculature. Such compounds may be administered in appropriate
pharmaceutical compositions either systemically or locally to
particular tissues or organs, in order to achieve the desired
systemic or local effect. Such desirable activities also include
induction of proliferation of endothelial cells, induction of
anti-apoptotic activity, induction of scatter activity, or any
combination of the foregoing activities. In a preferred embodiment,
any one of these activities by a compound of the invention is
reduced or inhibited in the presence of exogenous c-met
receptor.
[0030] Compounds of Formula I may be formulated into a suitable
pharmaceutical composition for delivery as appropriate, the
pharmaceutical composition containing at least one of the compounds
of Formula I together with a pharmaceutically-appropriate carrier,
excipient or diluent, or combinations of the foregoing. The
invention is also directed to pharmaceutical dosage forms
comprising an effective amount of a compound of Formula I for
administration on a periodic basis, such as four times a day, three
times a day, twice a day, and once a day, as well as less frequent
dosing, to provide an effective amount of the compound for its
intended pharmacological activity. Dosage forms administratable
orally, as well as parenteral dosage forms, are embraced
herein.
[0031] The invention is also directed to compounds of general
formula I: 2
[0032] wherein
[0033] R1 is SO.sub.2Alkyl, SO.sub.2-Aryl, CO-t-Butyl, COAryl,
CONHAlkyl; or CONHAryl; and
[0034] R3 is CHCH-heteroaryl; phenoxyphenyl; heteroaryl; or Aryl
substituted heteroaryl.
[0035] Non-limiting examples of compounds of Formula I include
[0036]
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone;
[0037] 1-(methylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole;
[0038]
2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-yl)propan-1-on-
e;
[0039]
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamide;
[0040]
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-1-yl)methanon-
e;
[0041]
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-py-
razol-1-yl)methanone;
[0042]
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-pyrazol-1-yl)methan-
one;
[0043]
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-1H-pyrazol--
1-yl)methanone;
[0044]
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamide;
[0045]
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thiazo-
l-5-yl)-1H-pyrazol-1-yl)methanone;
[0046] (3-benzhydryl-1H-pyrazol-1-yl)(4-chlorophenyl)methanone;
[0047]
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-carboxamid-
e;
[0048]
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-pyrazol--
1-yl)methanone;
[0049]
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl)phenoxy)benzonitr-
ile; and
[0050]
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole.
[0051] The invention is also directed to methods for preventing or
treating a disease in a mammal in which HGF/SF activity is
desirable comprising administering to the mammal an effective
amount of a pharmaceutical composition comprising a compound of
Formula I as described hereinabove. By way of non-limiting example,
conditions and diseases in which HGF/SF activity is desirable
include hepatic disease, ischemic and toxin-induced renal disease,
dysfunction in bone regeneration, poor hair growth, adverse wound
or tissue healing, and ischemic diseases. For example, augmenting
or restoring blood flow to ischemic tissues such as the heart
following myocardial infarction as well as in diabetes-related
ischemic conditions are amenable to treatment using the compounds
of the invention. The compounds are also useful in the treatment of
atherosclerosis, including reduction of lipid accumulation in the
vasculature. Such desirable activities also include induction of
proliferation of endothelial cells, induction of anti-apoptotic
activity, induction of scatter activity, or any combination of the
foregoing activities. In a preferred embodiment, any one of these
activities by a compound of the invention is reduced or inhibited
in the presence of exogenous c-met receptor.
[0052] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 depicts the stimulation of endothelial cell
proliferation by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone,
a compound of the invention with HGF/SF-like activity, and the
inhibition of the observed stimulation by inclusion of c-met.
[0054] FIGS. 2A-B show the induction of scatter of MDCK cells by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone.
[0055] FIG. 3 shows the protection of MDCK cells from
adriamycin-induced apoptosis by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]meth-
anone.
[0056] FIG. 4 shows a dose-response curve of the stimulation of
endothelial cell proliferation by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-
-1H-pyrazol-1-yl]methanone.
[0057] FIG. 5 shows improved blood flow in mice following removal
of the femoral artery after administration of a pharmaceutical
compositions comprising
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methan-
one.
[0058] FIG. 6 shows the stimulation of 3H-thymidine incorporation
into HUVEC by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanon-
e.
[0059] FIG. 7 depicts the phosphorylation of Erk by HGF/SF and
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone.
[0060] FIG. 8 demonstrates the efficacy of a pharmaceutical
composition comprising
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methan- one
in a pig wound healing model.
[0061] FIG. 9 shows the ability of a pharmaceutical composition
comprising
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
to increase capillary number in the ischemic mouse hindlimb.
[0062] FIG. 10 depicts the dose-dependent phosphorylation of HUVECs
and MDCK cells by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]met-
hanone.
[0063] FIG. 11 shows a positive effect on hindlimb ischemia in
diabetic mice by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
administration.
[0064] FIG. 12 demonstrates the reduction in lipid accumulation in
the aortas of Apo E-null mice as a result of treatment with
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone.
[0065] FIG. 13 shows that
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyraz- ol-1-yl]methanone
(also referred to herein as C6) prevents renal damage in a murine
model of renal ischemia.
[0066] FIG. 14 shows the results of a study in which
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
protected mice from mercuric chloride-induced renal damage.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The pharmaceutical compositions of the invention comprise
agents or compounds which modulate cellular proliferation to
provide new and effective agents for the prophylaxis and/or
treatment of various conditions and diseases in which limited
cellular proliferation is responsible for pathology, or in which
increased cellular proliferation is desirable in the prophylaxis or
treatment of the condition or disease. Such compounds may be
formulated into pharmaceutical compositions for administration to a
mammalian animal, preferably a human, for the treatment of a
condition or disease.
[0068] For example, the duration of wound healing, vascularization
of a damaged and/or ischemic organs, transplants or grafts,
normalization of myocardial perfusion as a consequence of chronic
cardiac ischemia or myocardial infarction, development or
augmentation of collateral vessel development after vascular
occlusion or to ischemic tissues or organs, and vascularization of
grafted or transplanted tissues, organs, or wound healing, may be
accelerated by promoting cellular proliferation, particularly of
vascular cells, by administration of a pharmaceutical composition
of the invention. Further utility is in the promotion of
endothelial growth in vascular grafts and transplants. Treatment of
diabetic ischemic disease is a further condition amenable to
treatment by the compounds of the invention. Pharmaceutical
compositions of the invention possess such activities.
[0069] As noted above, modulating cellular proliferation, either by
promoting the growth or new cells and/or formation of new blood
vessels is a therapeutically-desirable goal for the prophylaxis or
treatment of numerous conditions and diseases, including such major
pathologies as myocardial and other forms of ischemia, and wound
healing, as well as adjunctive therapy to increase the success rate
of, for example, organ transplants and skin grafts. The examples
provided herein below are merely illustrative of the range of
utilities of pharmaceutical compositions comprising proliferation
promoting agents, which include but are not limited to angiogenic
agents; such uses are known to the skilled artisan; moreover,
various citations referred to herein, and incorporated by
reference, offer guides to certain of the uses mentioned here.
[0070] Poorly perfused tissues and organs, such as the heart as a
sequela of myocardial infarction, other ischemic organs for example
as a result of diabetic macro- and microvasculopathy, as well as to
promote wound healing, organ transplantation, acceleration of
endothelial cell growth and vascularization of vascular grafts in
order to promote integration of the graft, prevent graft failure
due to reocclusion, and to enhance skin grafting, all of the
foregoing are exemplary of desirable targets for increasing
vascularization and uses of the angiogenic agents herein. Enhanced
vascularization of a chronically ischemic organ is a
therapeutically beneficial goal.
[0071] The term "angiogenesis," as used herein, refers to the
formation of blood vessels. Specifically, angiogenesis is a
multistep process in which endothelial cells focally degrade and
invade through their own basement membrane, migrate through
interstitial stroma toward an angiogenic stimulus, proliferate
proximal to the migrating tip, organize into blood vessels, and
reattach to newly synthesized basement membrane (see Folkman et
al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)). These
processes are controlled by soluble factors and by the
extracellular matrix (see Ingber et al., Cell, Vol. 58, pp. 803-805
(1985)).
[0072] The agents of the invention may be administered in
appropriate pharmaceutical compositions to the desired site in the
body or target tissue or organ by any means that achieves the
desired therapeutic effect. By way of non-limiting example,
proliferation promoting agents including angiogenic agents may be
administered locally, such as by injection or deposition of a
pharmaceutical composition comprising the agent in a target tissue
or organ, or by the implantation of a controlled release delivery
device or matrix containing the agent in a suitable pharmaceutical
composition, to achieve local effects. Such sites may be accessed
surgically, or via transcutaneous catheterization to gain access to
a tissue or organ through the major vasculature. For example,
enhancing the perfusion of the ischemic heart may be achieved by
use of a transcutaneous catheter that may be positioned to release
the angiogenic agent of the invention into the coronary
vasculature. Controlled and sustained release pharmaceutical
compositions and devices comprising the aforementioned agents are
fully embraced herein. Surgical or transcutaneous methods may also
be used. These and other means for contacting the agents of the
invention with the desired target cells, tissue or organs will be
readily apparent to the skilled artisan.
[0073] The present invention is directed to pharmaceutical
compositions of the above agents described in Formula I. The
formulation of the instant compounds in appropriate vehicles or
carriers or drug delivery systems is readily determinable by the
skilled artisan, and all such methods of delivery are embraced
herein. Examples are provided herein by way of illustration only,
and are not intended to be limiting whatsoever.
[0074] The pharmaceutical compositions of the invention may be for
administration for injection, or for oral, pulmonary, nasal or
other forms of administration. In general, comprehended by the
invention are pharmaceutical compositions comprising effective
amounts of a low molecular weight component or components, or
derivative products, of the invention together with
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such compositions include
diluents of various buffer content (e.g., Tris-HCl, acetate,
phosphate), pH and ionic strength; additives such as detergents and
solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants
(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
Thimersol, benzyl alcohol) and bulking substances (e.g. lactose,
mannitol); incorporation of the material into particulate
preparations of polymeric compounds such as polylactic acid,
polyglycolic acid, etc. or into liposomes. Hylauronic acid may also
be used. Such compositions may influence the physical state,
stability, rate of in-vivo release, and rate of in vivo clearance
of the present proteins and derivatives. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co.,
Easton, Pa. 18042) pages 1435-1712 which is herein incorporated by
reference. The compositions may be prepared in liquid form, or may
be in dried powder, such as lyophilized form.
[0075] Contemplated for use herein are oral solid dosage forms,
which are described generally in Remington's Pharmaceutical
Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at
Chapter 89, which is herein incorporated by reference. Solid dosage
forms include tablets, capsules, pills, troches or lozenges,
cachets or pellets. Also, liposomal or proteinoid encapsulation may
be used to formulate the present compositions (as, for example,
proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
Liposomal encapsulation may be used and the liposomes may be
derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).
A description of possible solid dosage forms for the therapeutic is
given by Marshall, K. In: Modern Pharmaceutics Edited by G. S.
Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated by
reference. In general, the formulation will include the component
or components (or chemically modified forms thereof) and inert
ingredients which allow for protection against the stomach
environment if necessary for stability, and release of the active
agent in the intestine.
[0076] Also specifically contemplated are oral dosage forms of the
above-derivatized component or components. The component or
components may be chemically modified so that oral delivery of the
derivative is efficacious. Generally, the chemical modification
contemplated is the attachment of at least one moiety to the
component molecule itself, where the moiety permits uptake into the
blood stream from the stomach or intestine. Also desired is the
increase in overall stability of the component or components and
increase in circulation time in the body. Examples of such moieties
include: polyethylene glycol, copolymers of ethylene glycol and
propylene glycol, carboxymethyl cellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and
Davis, 1981, "Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs,
Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y.,
pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4:185-189.
Other polymers that could be used are poly-1,3-dioxolane and
poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as
indicated above, are polyethylene glycol moieties.
[0077] For the component (or derivative) the location of release
may be the stomach, the small intestine (the duodenum, the jejunum,
or the ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment for acid-sensitive agents.
[0078] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0079] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic i.e. powder; for liquid
forms, a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0080] The therapeutic can be included in the formulation as fine
multi-particulates in the form of granules or pellets of particle
size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression.
[0081] Colorants and flavoring agents may all be included. For
example, the compound may be formulated (such as by liposome or
microsphere encapsulation) and then further contained within an
edible product, such as a refrigerated beverage containing
colorants and flavoring agents.
[0082] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans and starch. Certain inorganic salts may
be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and
Avicel.
[0083] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates include but are not limited to starch, including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0084] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0085] An anti-frictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0086] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0087] To aid dissolution of the therapeutic into the aqueous
environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential non-ionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the agent either
alone or as a mixture in different ratios.
[0088] Additives which potentially enhance uptake of the agent are
for instance the fatty acids oleic acid, linoleic acid and
linolenic acid.
[0089] Controlled release oral formulation may be desirable. The
agent could be incorporated into an inert matrix which permits
release by either diffusion or leaching mechanisms, e.g., gums.
Slowly degenerating matrices may also be incorporated into the
formulation. Some enteric coatings also have a delayed release
effect.
[0090] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The therapeutic agent could also be given in a film coated
tablet and the materials used in this instance are divided into 2
groups. The first are the nonenteric materials and include methyl
cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose,
providone and the polyethylene glycols. The second group consists
of the enteric materials that are commonly esters of phthalic
acid.
[0091] A mix of materials might be used to provide the optimum film
coating. Film coating may be carried out in a pan-coater or in a
fluidized bed or by compression coating.
[0092] Also contemplated herein is pulmonary delivery of the
present agent. The agent is delivered to the lungs of a mammal
while inhaling and traverses across the lung epithelial lining to
the blood stream. Other reports of this include Adjei et al., 1990,
Pharmaceutical Research, 7:565-569; Adjei et al., 1990,
International Journal of Pharmaceutics, 63:135-144 (leuprolide
acetate); Braquet et al., 1989, Journal of Cardiovascular
Pharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al.,
1989, Annals of Internal Medicine, Vol. III, pp. 206-212
(al-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146
(a-1-proteinase); Oswein et al., 1990, "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery
II, Keystone, Colo., March, (recombinant human growth hormone);
Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-gamma and
tumor necrosis factor alpha) and Platz et al., U.S. Pat. No.
5,284,656 (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong
et al.
[0093] Intravenous or other injectable forms of the compounds of
the invention are also embraced herein, wherein the dosage form
includes the agent in a suitable solution or reconstitutable form
for injection.
[0094] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0095] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler
powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
[0096] All such devices require the use of formulations suitable
for the dispensing of the agent. Typically, each formulation is
specific to the type of device employed and may involve the use of
an appropriate propellant material, in addition to the usual
diluents, adjuvants and/or carriers useful in therapy. Also, the
use of liposomes, microcapsules or microspheres, inclusion
complexes, or other types of carriers is contemplated.
[0097] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise protein (or derivative)
dissolved in water at a concentration of about 0.1 to 25 mg of
agent per mL of solution. The formulation may also include a buffer
and a simple sugar (e.g., for protein stabilization and regulation
of osmotic pressure). The nebulizer formulation may also contain a
surfactant.
[0098] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the agent
suspended in a propellant with the aid of a surfactant. The
propellant may be any conventional material employed for this
purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0099] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing the agent and
may also include a bulking agent, such as lactose, sorbitol,
sucrose, or mannitol in amounts which facilitate dispersal of the
powder from the device, e.g., 50 to 90% by weight of the
formulation. The agent should most advantageously be prepared in
particulate form with an average particle size of less than 10 mm
(or microns), most preferably 0.5 to 5 mm, for most effective
delivery to the distal lung.
[0100] Nasal delivery of the agent is also contemplated. Nasal
delivery allows the passage of the agent to the blood stream
directly after administering the therapeutic product to the nose,
without the necessity for deposition of the product in the lung.
Formulations for nasal delivery include those with dextran or
cyclodextran.
[0101] For controlled delivery, incorporation of the material into
particulate preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, etc. or into liposomes may be used, or the
use of a controlled release device, such as an implantable osmotic
or other type of pump. Another form of a controlled release of this
therapeutic is by a method based on the Oros therapeutic system
(Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane
which allows water to enter and push drug out through a single
small opening due to osmotic effects. Likewise, the skilled artisan
will be amply aware of suitable delivery methods that may be
extended to the agents of the invention to achieve the intended
therapeutic goals of the invention.
[0102] The compounds of the invention may be delivered by
intravenous, intraarterial, intraperitoneal, intramuscular, or
subcutaneous routes of administration. A pharmaceutical composition
for parenteral administration may be prepared using one or more of
the compounds of Formula I, with a suitable diluent, excipient, or
other components, to provide a stable and effective formulation for
injection.
[0103] A subject in whom administration of a pharmaceutical
composition of the invention in an effective therapeutic regiment
for a disease or condition exemplified above, but not so limiting,
is preferably a human, but can be any animal. Thus, as can be
readily appreciated by one of ordinary skill in the art, the
methods and pharmaceutical compositions of the present invention
are particularly suited to administration to any animal,
particularly a mammal, and including, but by no means limited to,
domestic animals, such as feline or canine subjects, farm animals,
such as but not limited to bovine, equine, caprine, ovine, and
porcine subjects, wild animals (whether in the wild or in a
zoological garden), research animals, such as mice, rats, rabbits,
goats, sheep, pigs, dogs, cats, etc., avian species, such as
chickens, turkeys, songbirds, etc., i.e., for veterinary medical
use.
[0104] For all of the above molecules, as further studies are
conducted, information will emerge regarding appropriate dosage
levels for treatment of various conditions in various patients, and
the ordinary skilled worker, considering the therapeutic context,
age and general health of the recipient, will be able to ascertain
proper dosing. The appropriate effective dosage of an agent of the
invention may be readily determinable by following standard
methods. Several animal models are described herein which model
conditions and diseases encountered in the clinical setting, and as
part of a drug development process, efficacious doses in animal
studies, in particular, dose-response studies, are translated into
appropriate doses for testing in humans, by following guidelines
well known to those skilled in the art. Thus, an effective dose in
a human may be determined following such industry-standard
guidelines. As shown in the Examples, infra, a dosage approximately
equal to the equivalent of 0.1 mg/kg/day to about 10 mg/kg/day, and
more preferably, from about 0.5 mg/kg/day to about 5 mg/kg/day, is
therapeutically effective in the disease model. Generally, for
intravenous injection or infusion, or with a derivative of the
component, dosage may be lower. The dosing schedule may vary,
depending on the circulation half-life, and the formulation
used.
[0105] As mentioned above, pharmaceutical compositions comprising
the proliferation promoting agents including angiogenic agents of
the invention may be used to promote endothelial cell and
microvessel growth, with the goal of increasing vascularization and
perfusion of tissues and organs in the body. They may also be used
to promote growth of other cells types, such as those expressing
c-met. Also as noted above, the agents may be locally applied or
delivered to the desired site or sites. While the invention
embraces any and all uses of angiogenic an generally proliferation
promoting agents for humans and other mammals, some examples
include treatment of ischemic tissues and organs, such as after
injury, including myocardial damage after a heart attack, promoting
vascularization of transplanted, reattached or translocated tissues
or organs, such as following organ transplants, traumatic injury,
promotion of wound healing, skin and other organ grafting, to name
some examples. They are particularly useful for promoting the
growth of endothelial cells in vascular grafts and transplants.
[0106] With regard to the compounds of Formula I herein, as used
herein, the term "alkyl" means straight-chain, branched-chain or
cyclo saturated aliphatic hydrocarbon groups preferably containing
from one to about 6 carbon atoms. Representative of such
straight-chain groups are methyl, ethyl, butyl, pentyl, hexyl and
the like. Examples of branched-chain groups include isopropyl,
isobutyl and t-butyl. Cycloalkyl includes groups such as but not
limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The
term "aryl" refers to, for example, phenyl, biphenyl and naphthyl
groups, which are optionally substituted by one or more halogen (F,
Cl, Br and I), C1 to C4 alkyl, or C1 to C4 alkyloxy, where alkyloxy
refers to an alkyl group as defined above attached to the remainder
of the molecule by oxygen. Examples of alkyloxy include methoxy,
ethoxy, propoxy, isopropoxy and the like. The term "heteroaryl"
refers to heterocyclic groups containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen
and sulfur. Examples include but are not limited to isoxazolyl,
phenylisoxazolyl, furyl, pyrimidinyl, quinolyl, tetrahydroquinolyl,
pyridyl, imidazolyl, pyrrolidinyl, 1,2,4-triazoylyl, thiazolyl,
thienyl, and the like. The aryl or heteroaryl group may be
optionally substituted by one or more halogen (F, Cl, Br and I), C1
to C4 alkyl, C1 to C4 alkyloxy as described above, trifluoromethyl,
difluoromethyl, nitro, hydroxy, amine (optionally alkyl
substituted), or another aryl or another heteroaryl group as
described above.
[0107] The invention is directed to a pharmaceutical composition
comprising a compound that modulates HGF/SF activity with the
general formula I: 3
[0108] wherein
[0109] R1 is SO.sub.2Alkyl, SO.sub.2-Aryl, CO-t-Butyl, COAryl,
CONHAlkyl, or CONHAryl, and
[0110] R3 is CHCH-heteroaryl; phenoxyphenyl; heteroaryl; or Aryl
substituted heteroaryl;
[0111] and a pharmaceutically-acceptable carrier, excipient or
diluent.
[0112] Preferably, R1 may be SO.sub.2Alkyl, wherein Alkyl is C1 to
C4 straight-chained, branched or cyclo, most preferably
SO.sub.2CH.sub.3; SO.sub.2-Aryl, wherein Aryl is halo, C1-4 alkyl
or alkyloxy substituted phenyl; COAlkyl, wherein alkyl is C1 to C6
straight-chained alkyl, branched alkyl or cycloalkyl, most
preferably CO-t-Butyl; COAryl wherein Aryl is phenyl substituted
with halo, C1-C4 alkyl or alkyloxy; CONHAlkyl wherein alkyl is C1
to C6 straight-chained alkyl, branched alkyl or cycloalkyl, most
preferably CONHCH.sub.3; or CONHAryl, wherein aryl is phenyl
substituted with halo, C1 to C4 alkyl or C1 to C4 alkyloxy. R3 may
be CHCH-heteroaryl, where in heteroaryl includes but is not limited
to both cis and trans CHCH-3-thienyl, CHCH-2-furyl and
CHCH-3-furyl, and substituted CHCH-thienyl and CHCH-furyl, most
preferably CHCH-2-thienyl; phenoxyphenyl; heteroaryl; or aryl
substituted heteroaryl.
[0113] The invention includes pharmaceutical compositions
comprising a compound of Formula I. Certain of the compounds of
Formula I are novel, and the present invention is also inclusive of
pharmaceutical compositions comprising all such novel compounds.
Moreover, the invention is also directed to a pharmaceutical
composition comprising at least one compound of Formula I, in a
pharmaceutically-acceptable carrier, for any of the uses described
herein.
[0114] The invention is also directed to compounds of Formula I.
These pyrazoles may be synthesized by several methods. In method A,
illustrated below, 3-bromopyrazole (commercially available) will
under go alkylation or acylation by reaction with an acyl chloride
or alkyl halide in a suitable polar aprotic solvent such as
dichloromethane and base such triethylamine to form intermediate 2.
Coupling reaction with a boronic acid, using a palladium catalyst,
base such as K2CO3 or CsCO3, and warming in an aprotic solvent such
as benzene, toluene, or xylene will provide target pyrazoles 3.
Alternatively, 3-bromopyrazole 2 may be coupled with vinyl
compounds in the presence of a catalyst prepared in situ from 2.5%
Pd(AcO)2 with 5% m.sulfonated triphenylphosphine (TPPTS) in an
aqueous solvent such as water and ethanol. (Ref. Genet, J. P.,
Blart, E.; Savignac, M., Synlett, 1992, 715-717).
[0115] For example, using Method A, below, for the preparation of
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
(also referred to herein as C6), one may react 3-bromopyrazole with
4-chlorobenzoyl chloride (commercially available, for example, from
Aldrich Chemical Co., Milwaukee, Wis.) to provide the first
intermediate in Method A. Then reaction of this material with
E-2-(thienylethenyl)boro- nic acid (commerically available, for
example, from Combi-Blocks Inc., San Diego, Calif.) forms the
desired product. Other compounds may be likewise prepared from the
corresponding benzoyl chlorides as well an many different types of
boronic acid analogs that are commercially available or readily
synthesized. 4
[0116] In Method B, depicted below, cinnamaldehydes undergo
reaction with hydrazides to form hydrazone intermediates. The
dianion of the corresponding hydrazones undergo reaction with
esters, amides, acid anhydrides, acid chlorides and alkyl
carbonates to form target pyrazoles. (ref. Tetrahedron Lett. 1983,
24(31), 3239-3242. 5
[0117] The foregoing are merely exemplary of synthetic routes to
these compounds.
[0118] These compounds generally exhibit HGF/SF stimulatory or
agonist activity. Non-limiting examples of compounds of Formula I,
with formulae and molecular weights, include
1 Molecular Chemical name Formula weight
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-
C.sub.16H.sub.11ClN.sub.2OS 314.80 yl]methanone (also referred to
herein as C6) 1-(methylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazol-
e C.sub.10H.sub.10N.sub.2O.sub.2S.sub.2 254.33
2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-
C.sub.14H.sub.16N.sub.2OS 260.36 yl)propan-1-one
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-
C.sub.11H.sub.11N.sub.3OS 233.29 carboxamide
(4-chlorophenyl)(3-(3-phenylisoxazol-5-y- l)-1H-
C.sub.19H.sub.12ClN.sub.3O.sub.2 349.78 pyrazol-1-yl)methanone
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-
C.sub.20H.sub.13Cl.sub.2N.sub.3O.sub.2 398.25
methylisoxazol-4-yl)-1H-pyrazol-1-yl)methanone
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-
C.sub.18H.sub.11ClN.sub.- 2OS.sub.2 370.88 pyrazol-1-yl)methanone
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-
C.sub.20H.sub.10Cl.sub.2F.sub.2N.sub.2O.sub.2 419.22
1H-pyrazol-1-yl)methanone N-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyraz-
ole-1- C.sub.16H.sub.13N.sub.3OS 295.37 carboxamide
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-
C.sub.22H.sub.14ClN.sub.3OS.sub.3 468.02 methyl-1,3-thiazol-5-yl)--
1H-pyrazol-1-yl)methanone (3-benzhydryl-1H-pyrazol-1-yl)4-
C.sub.23H.sub.17ClN.sub.2O 372.86 chlorophenyl)methanone
N-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-
C.sub.16H.sub.12ClN.sub.3OS 329.81 1-carboxamide
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-
C.sub.18H.sub.13ClN.sub.4O 336.78 yl)-1H-pyrazol-1-yl)methanone
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-
C.sub.23H.sub.13Cl.sub.2N.sub.3O.sub.2 434.28
yl)phenoxy)benzonitrile 1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thien-
yl)vinyl)-1H- C.sub.15H.sub.11ClN.sub.2O.sub.2S.sub.2 350.85
pyrazole
[0119] The invention is also directed to novel compounds of Formula
I, above.
[0120] The invention is also directed to methods for treating a
mammalian animal for a disease or condition described above by
administering to the animal an effective amount of a compound of
Formula I, described hereinabove, to achieve the intended
purpose.
[0121] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
EXAMPLE 1
Pharmaceutical Compositions
[0122] A n oral dosage form of a compound of Formula I may be
provided as follows:
2 Compound of Formula I 50 mg Magnesium stearate 10 mg Sorbitol 200
mg
[0123] A solution for intravenous administration may be prepared
from:
3 Compound of Formula I 10 mg Saline 100 cc
EXAMPLE 2
HGF/SF-Like Cellular Proliferative Activity of a Compound of the
Invention
[0124] Using the endothelial cell proliferation assay described
above, the compound
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanon- e
was shown to increase HUVEC proliferation by two to five fold. The
specificity of the stimulation of endothelial cell growth by the
compound as measured by .sup.3H-thymidine incorporation was tested
by pre-incubation of cells with the HGF/SF receptor c-met. In FIG.
1, the first bar represents control cells; the second bar
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
at 6 microgram/ml; and the third bar:
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-- 1H-pyrazol-1-yl]methanone
at 6 microgram/ml plus c-met receptor, 100 microgram/ml.
(4-Chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]met- hanone
by itself stimulated .sup.3H-thymidine incorporation by 84%. Thus,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
is as effective as HGF/SF in stimulating HUVEC proliferation. In
the presence of c-met, the
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]met- hanone
stimulation of .sup.3H-thymidine incorporation was inhibited by
75%. In another related experiment,
(4-chlorophenyl)[3-(2-(2-thienyl)viny- l)-1H-pyrazol-1-yl]methanone
(12 microgram/ml) was incubated with the initial target molecule
C-met receptor (5 microgram/ml) for 30 min and then added to the
cells. Compound-induced EC proliferation was blocked by 40% in the
presence of C-met receptor.
EXAMPLE 3
Scatter of MDCK Cells
[0125]
(4-Chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
was further tested for HGF/SF activity in a standard scatter assay
that is specific for HGF/SF. The ability to scatter was
demonstrated for the first time using a non-peptide candidate
compound. Scatter of MDCK cells by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
further demonstrates that its actions are mediated through
stimulation of the c-met receptor. As shown in FIG. 2, the compound
caused scattering of MDCK cells similar to that seen with HGF/SF.
FIG. 2A: Control cells; FIG. 2B:
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1yl]methanone, 6
microgram/ml.
EXAMPLE 4
Anti-Apoptotic Activity of
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyra-
zol-1-yl]methanone
[0126] HGF/SF has significant anti-apoptotic activity in a number
of cultured cell lines. Using the MTT cell viability assay the
ability of
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
to protect cells from adriamycin-induced apoptosis was evaluated.
Like HGF/SF,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
was able to significantly block adriamycin-induced apoptosis in
MDCK cells (FIG. 3). Cell viability was unchanged by either HGF/SF
alone (column 2),
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]metha- none
alone (column 5) or HGF/SF and
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl- )-1H-pyrazol-1-yl]methanone
combined (column 7). Adriamycin (15 mM) decreased cell viability to
56% of control (column 3). Treatment with either HGF/SF (column 4)
or (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-py-
razol-1-yl]methanone (column 6) effected nearly complete (94%)
protection from adriamycin-induced apoptosis.
[0127] In another cell line, 90% protection was afforded by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone.
EXAMPLE 5
Effect of
(4-chlorophenyl)(3-2-(2-thienyl)vinyl)-1H-pyrazol-1-yl-methanone on
HUVEC Proliferation
[0128] FIG. 4 shows a dose-response relationship between the level
of level
(4-chlorophenyl)(3-2-(2-thienyl)vinyl)-1H-pyrazol-1-yl-methanone
and HUVEC proliferation.
EXAMPLE 6
In vivo Blood Flow Improvement Assay
[0129] FIG. 5 shows the results of an in-vivo experiment in which a
pharmaceutical composition comprising
(4-chlorophenyl)[3-(2-(2-thienyl)vi- nyl)-1H-pyrazol-1-yl]methanone
was administered to mice for seven days following removal of the
femoral artery. The results show significant improvement in blood
flow with the compound.
EXAMPLE 7
Other Compounds with Activities
[0130] In a similar manner as described above, the following
compounds related were evaluated for stimulation of endothelial
cell proliferation. Three different rounds of testing were
performed.
4 Stimulation/Inhibition Stimulation/Inhibition Compound at 5
micrograms/ml at 10 micrograms/ml
1-(methylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H- 74% stimulation Not
significant pyrazole 2,2-dimethyl-1-(3-(2-(2-thienyl)vinyl)- -1H-
No effect 55% stimulation pyrazole-1-yl)propan-1-one
N-methyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole- 70% stimulation 42%
stimulation 1-carboxamide (4-chlorophenyl)(3-(3-phenylisoxa-
zol-5-yl)- 54% stimulation 60% stimulation
1H-pyrazol-1-yl)methanon- e
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)- 40% stimulation 40%
stimulation 1H-pyrazol-1-yl)methanone
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H- 40% stimulation Not
significant pyrazole-1-carboxamide
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2- 20% stimulation 33%
stimulation thienyl)-4-methyl-1,3-thiazol-5-yl- )-1H-
pyrazol-1-yl)methanone (3-benzhydryl-1H-pyrazol-1-yl)(- 4- No
effect 25% stimulation chlorophenyl)methanone
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)- No effect 55%
stimulation 1H-pyrazole-1-carboxamide
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-- pyrazol- 60% stimulation 90%
stimulation 5-yl)phenoxy)benzonitrile
EXAMPLE 8
Stimulation of Endothelial Cell Proliferation by
(4-chlorophenyl)[3-(2-(2--
thienyl)vinyl)-1H-pyrazol-1-yl]methanone
[0131] One of the compounds identified with HGF/SF-like activity,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone,
was able to stimulate endothelial cell proliferation in vitro (FIG.
6: first bar: control cells; second bar
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-
-pyrazol-1-yl]methanone, 38 mM; third bar: HGF/SF, 20 ng/ml; fourth
bar:
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone+HGF/SF)-
. The specificity of the stimulation by this compound of growth of
HUVECs by .sup.3H-thymidine incorporation was tested by
pre-incubation of cells with the HGF/SF receptor c-met.
(4-Chlorophenyl)[3-(2-(2-thienyl)vinyl)-1- H-pyrazol-1-yl]methanone
by itself stimulated .sup.3H-thymidine incorporation by more than 5
fold (FIG. 18, bar 2).
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
is as effective as HGF/SF in stimulating HUVEC proliferation. In
the presence of c-met the
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]meth- anone
stimulation of .sup.3H-thymidine incorporation was inhibited by
75%. Scattering of MDCK cells in culture is a known specific effect
of scatter factor and
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methan- one
also has this ability, the first demonstration of this activity in
a non-peptide compound.
(4-Chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol--
1-yl]methanone, like HGF/SF, did not stimulate the growth of
fibroblast cell lines and both showed similar inhibitory effects in
HepG2 hepatoma cell lines.
EXAMPLE 9
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
Causes Phosphorylation of c-met and Erk
[0132] Using immunoprecipitation and Western blotting we were able
show that like HGF/SF,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl- ]methanone
causes phosphorylation of the signaling protein Erk (FIG. 7). Both
HGF/SF (lane 4) and
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyraz- ol-1-yl]methanone
(lane 2) showed significant amounts of phosphorylated Erk compared
to unstimulated control cells (lane 1). A small molecule
antagonist,
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(3-
-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl)methanone had no effect
on phosphorylation of Erk (Lane 3). Total Erk is shown on the
bottom.
EXAMPLE 10
Wound Healing Studies
[0133] The angiogenic properties of a pharmaceutical composition
comprising
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methan- one
were further tested in a pig model of wound healing. Full thickness
8-mm skin wounds were produced in pigs and five days later the
wounds were excised, stained with H&E and blood vessels counted
in five areas from each section under high power. Wounds treated
with
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
(500 micrograms) demonstrated a 33% greater density of blood
vessels compared to vehicle-treated (DMS) controls (FIG. 8).
EXAMPLE 11
Increase in Capillary Numbers by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1-
H-pyrazol-1-yl]methanone
[0134] Mice were subjected to unilateral hindlimb ischemia and
treated with either the HGF/SF agonist,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1- H-pyrazol-1-yl]methanone
(25 micrograms/day) or vehicle for either two or three weeks prior
to sacrifice. Hindlimb muscles were frozen in liquid nitrogen and
capillaries stained by the alkaline phosphatase technique and the
number of capillaries per muscle fiber counted in 6 to 12 random
areas of the muscle by a blinded observer.
[0135] Recovery in mice with hindlimb ischemia by increasing the
number of capillaries in the ischemic muscle was observer (FIG. 9).
At 2 weeks there was a 42% greater number of capillaries per muscle
fiber in
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone-treated
mice compared to vehicle treated controls. This increased number of
capillaries persisted at 3 weeks, the last time point for which
samples were analyzed.
EXAMPLE 12
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
Produces a Dose-Dependent Phosphorylation of c-met
[0136] Studies of c-met phosphorylation have been extended to
demonstrate that the phosphorylation is dose-related and occurs in
both HUVECs as well as MDCK cells (FIG. 10). HUVECs (left set) or
MDCK cells (right set) were treated with either HGF/SF or
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl-
)-1H-pyrazol-1-yl]methanone, solubilized lysates were prepared from
cells and immunoprecipitation of phosphorylated c-met and total
c-met using specific antibodies was performed using standard
techniques. Immunoprecipitates were separated on SDS-polyacrylamide
gels and proteins were transferred to nitrocellulose membranes and
detection of phosphorylated (top) and total c-met (bottom) was
performed using an ECL chemiluminescence system (Amersham). Both
HGF/SF and
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
showed significant amounts of phosphorylated c-met compared to
unstimulated control cells. Total c-met is shown on the bottom.
[0137] This result further substantiates the findings that, like
HGF/SF,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
produces its effects through activation of the c-met receptor.
EXAMPLE 13
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
Reduces Hindlimb Ischemia in NOD Mice
[0138] Female non-obese diabetic mice (NOD) were subjected to
hindlimb ischemia by removal of the left femoral artery under
ketamine/xylazine anesthesia. Mice were injected daily for four
weeks with either
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
(25 micrograms) or vehicle (RPMI 1640+1% BSA). Hindlimb blood flow
was measured using a Laser Doppler imager 7 days later. In FIG. 11,
the y-axis represents the ratio of flux in the left, ischemic limb
to flux in the right, non-ischemic limb. At 7 days mice injected
with
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
(n=7) showed recovery to 59.+-.6% of the non-ischemic side compared
to 32.+-.6% for control animals injected with vehicle (n=8)
(P<0.01).
EXAMPLE 14
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
Decreases Neutral Lipid Accumulation in ApoE-Null Mice
[0139] Eight-week old Female ApoE-null mice were injected daily for
four weeks with either
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl- ]methanone
(25 micrograms) or vehicle (RPMI 1640+1% BSA). The abdominal aortas
were then removed and fixed in 10% formalin prior to staining with
Oil Red O to visualize neutral lipids. Stained aortas were mounted
on slides and scanned with a Hewlett Packard ScanJet 4c scanner.
Dark red stained neutral lipids were measured using a computerized
image analysis system (Universal Imaging Corp). Results are
presented in FIG. 12 as the percentage of the aorta stained dark
red. (4-Chlorophenyl)[3-(2-(2-thieny-
l)vinyl)-1H-pyrazol-1-yl]methanone treatment resulted in a
significant decrease in lipid accumulation to 30.+-.1% compared to
85.+-.3% in vehicle treated mice (P<0.05).
EXAMPLE 15
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
Prevents Increased Creatinine by Renal Ischemia
[0140] Male C57BL/6 mice were anesthetized with ketamine/xylazine
and the left renal vessels were occluded with a clamp for 30
minutes. Following release of the occlusion, the right kidney was
removed and the mouse was sutured closed. Mice were injected daily
with either
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
(25 micrograms) or vehicle (RPMI 1640.+-.1% BSA) and blood samples
collected over a period of 1 week for creatinine measurements to
assess the renal damage in response to ischemia. As shown in FIG.
13,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
treatment prevented the initial large increase in creatinine that
was observed in vehicle treated mice at day 1.
EXAMPLE 16
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
Protects Against Mercury-Induced Renal Damage
[0141] Mercuric chloride (HgCl.sub.2) was administered
intraperitoneally (i.p.) to mice at a dosage of 7 mg/Kg body
weight. Immediately thereafter,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]metha- none
(25 micrograms/mouse) and control (DMSO) solution were given i.p.
and continued daily for 3 days. Blood samples were harvested before
the experiment and on the 2nd and 4th day after the HgCl.sub.2
injection. Mice were sacrificed on the 4th day. Kidneys were
collected for histology and molecular biology.
[0142] FIG. 14 shows protection of kidneys from functional
impairment by mercuric chloride. Untreated animals' renal function
declined as shown by an increasing serum creatinine level. In
contrast, treatment of animals with
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
prevented the impairment.
[0143] The present invention is not to be limited in scope by the
specific embodiments describe herein. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
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* * * * *